In our recent work, we have shown that Gamma-glutamyltransferase (GGT), a membrane-bound enzyme, is completely and irreversibly inactivated by thiobarbituric acid and phenobarbital. Both the compounds were shown to be active-site directed inhibitors of GGT. Also, the stability of the isolated [3H]-phenobarbital-GGT complex under rigorous denaturation conditions provided compelling evidence that phenobarbital is covalently bound to GGT. Little is known, to date, regarding the active-site residue(s) in GGT that are essential for the catalytic process. The purpose of this research proposal is to initiate studies which will extend and amplify our initital observations to gain further insights into the nature of the active-site residues essential for enzymic catalysis. For the proposed studies, a large batch of GGT from rat kidneys will be purified. Studies on the inactivation of GGT by other recently discovered inhibitors of GGT, i.e., alloxan, 4,6-dihydroxy-2-methylmercapto pyrimidine, 2,4-dihydroxy-5-thio-pyrimidine and uracil will be continued. Identification of active-site amino acid residue(s) that are reactive to some selected pyrimidine inhibitors (e.g., phenobarbital and 2-S-methylthiobarbituric acid) and other known irreversible inactivators of GGT such as 6-diazo-5-oxo-L-norleucine (DON) and (AlphaS, 5S)-Alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) will be carried out. For these studies, the purified GGT will be treated with an excess of radiolabeled-inhibitor for complete inactivation of the enzyme activity. For identification of chemically-modified residues, the separated labeled small subunit of GGT will be subjected to controlled proteolysis using selective and specific proteolytic enzyme(s). The radiolabeled peptide(s) will be purified using techniques such as high performance liquid chromatography, high voltage electrophoresis, ion-exchange and gel filtration chromatography. The sequence of purified radiolabeled peptides(s) will be determined using automated Edman degradation method. Subsequently, the chemically-modified residue(s) will be identified using an amino-acid analyzer. These studies are essential to understand the active-site residue(s) in GGT that are responsbile for the catalytic process and to understand the mechanism of GGT action. Also, these studies may aid in developing specific irreversible inhibitors of GGT that are active in vivo. This, in turn, may help understand the physiological role of GGT.